In vitroSodium Fluoride Treatment Significantly Affects Apoptosis and Proliferation in the Liver of Embryonic Chickens

Abstract

Sodium fluoride (NaF), although helpful in preventing dental decay, may negatively affect the body. The aim of this study was to examine the effects of a 6-hin vitrotreatment of livers isolated from 14-day-old chicken embryos with NaF at doses of 1.7 (D1), 3.5 (D2), 7.1 (D3) and 14.2 mM (D4), with regard to apoptosis, cell proliferation and tissue structure. The mRNA expression of the apoptosis regulatorsCYCS,APAF1,BCL2,CASP3,CASP9andTMBIM1was analysed by the qPCR method. Apoptotic cells were detected by a TUNEL assay. The tissue and DNA structure were also analysed by histological staining (H&E, Feulgen). The number of proliferating cells was determined and the apoptosis regulatory proteins were localised by the immunohistochemical staining of PCNA, CASP3 and APAF1. The results showed that the mRNA expression ofCYCS,BCL2,CASP3,CASP9andAPAF1increased significantly in the D1 group, as did that ofCASP9in the D3 group and ofBCL2andAPAF1in the D4 group. The number of apoptotic cells increased significantly in the D4 group, where they increased from 18% to 49%. On the other hand, the number of proliferating cells decreased gradually, in a dose-dependent manner, from 84% in the control group to 5.5% in the D4 group. The expression of apoptosis-regulating factors also increased: in the D3 and D4 groups, the CASP3 immunopositive reaction was more intensive in single cells in the embryonic livers, whereas that of APAF1 increased in the hepatocytes as well as in the hepatic blood vessel walls. The mechanism of the effect of NaF on apoptosis in the embryonic liver is very complex. In the groups exposed to higher doses of NaF, apoptosis was significantly stimulated, while proliferation was inhibited and the tissue structure was damaged. The expression of apoptosis regulators at the mRNA and protein levels increased, but the mRNA expression did not depend on the NaF dose. These results reveal that NaF, by changing the balance between apoptosis and the proliferation of hepatocytes, may disturb the development and function of the liver in embryonic chickens. Therefore, the risk of exposure to NaF should be considered when determining the standards for human and animal exposure to this compound.